1. Field of the Invention
This invention relates to a method of purifying proteins and nucleic acids
2. Background of the Invention
One of the largest contributors to false positive and false negative results in rapid detection technologies is inconsistent, variable processed samples. Sample preparation represents a critical, yet under developed capability. Present day sample preparation is usually performed manually. When automated, sample preparation involves mechanizing manual processing methods resulting in large, complex, robotic systems with significant reagent/waste streams and consumable usage.
Generic methods for the purification of DNA from cells or mixtures of cells have been available for many years and include alcohol precipitation, silica binding, standard gel electrophoresis methods, and phenol/chloroform extraction. The polymerase chain reaction (PCR), through its use of primers to amplify and detect regions of the previously-purified nucleic acid specific for different classes of organisms. The ability to develop generic purification methods for nucleic acids is due largely to the fact that all nucleic acid molecules are similar in chemical structure and these similarities can be taken advantage of when developing purification methods. However, most of these methods are time consuming and must currently be performed manually.
Proteins, on the other hand, are very different in structure from one type of protein to the next. Therefore, protein purification has largely focused on separation methods based on unique protein characteristics such as differences in size, charge, hydrophobicity, isoelectric point, antibody binding and/or enzyme-substrate specificities. When a number of these methods are performed in tandem eventually leads to a protein that has been purified away from all other proteins in the starting mixture. Typically, separation methods include chromatography electrophoresis, immunoprecipitation and magnetic separation techniques and the like. Chromatography is performed using columns which are typically quite large and require expensive equipment to obtain and analyze the samples. Therefore, methods currently used to purify nucleic acids are not used to purify protein and conversely, methods used to purify protein are not used purify nucleic acids.
Isotachophoresis is a technique used to separate charged particles using a discontinuous electrical field to create sharp boundaries between the sample constituents. In this method, the sample is introduced between a fast leading electrolyte and a slow moving electrolyte to create a window between which a subset of constituents from a complex matrix can be segregated from other matrix constituents. The segregation occurs after application of an electrical field to the sample and the electrolytes are allowed to separate by charge. The electrolytes used for a separation are selected experimentally so that as many contaminating constituents as possible are excluded from the final sample.
Isotachophoresis is currently used in the field of protein purification as one potential method in capillary electrophoresis for the separation of specific proteins from a mixture of proteins. However, one disadvantage of isotachophoresis in capillary use is that one can only purify either negative or positively charged ions in the capillary tubes because these moieties will migrate in different directions upon application of the electrical field. The most common use of isotachophoresis is in protein stacking gels where protein samples are added to a gel in a wide band for separation by molecular weight. However, before separation by molecular weight can occur, the protein must be concentrated into a small band to increase resolution.
U.S. Pat. No. 3,869,365 (Sunden) is drawn to a broad method of counter-flow isotachophoresis comprising two electrolytes and the flow and voltage adjusted to maintain the sample at a desired position in the column. U.S. Pat. No. 3,948,753 (Arlinger) is drawn to an apparatus for isotachophoresis comprising a column, capillary tube, a detector, and a shunt tube bifurcating the column. U.S. Pat. No. 6,685,813 B2 (Williams et al.) is drawn to a method of separating components using isotachophoresis. U.S. Appl. Ser. No. 2004/0060821 A1 (Williams et al.) is drawn to a method of separating components using isotachophoresis. Application Ser. No. WO 2008/025806 (Gerhard Weber) is directed to a method of separating particles using electrophoresis and discloses isotachophoresis. Baumann, G. and Chrambach, A. disclosed the use of isotachophoresis for the isolation of hormones. (Proc. Natl. Acad. Sci. USA, 73(3): 732-736, (1976)). Böttcher, A. et al. disclosed preparative isotachophoresis for separation of human plasma lipoproteins, apolipoproteins and HDL subfractions, (J. Lipid Res. 41:905-915, (2000)). None disclose the simultaneous separation and purification of both nucleic acids and proteins. U.S. application Ser. No. 2004/0031683 A1 (Eipel el al.) is drawn to a method of fractionating proteins using several procedures, including isotachophoresis. The two-step process includes capillary electrophoresis, but does not disclose the separation of nucleic acids.
Application Ser. No. EP 05076569.2 (Stichting voor de Technische Wetenschnappen) is drawn to a device for separating particles and the use of isotachophoresis for the non-simultaneous separation of nucleic acids or proteins and the use of binding molecules. Blessum, C. et al. disclose capillary electrophoresis in the separation of proteins, nucleic acids and lipoproteins, and its use in isotachophoresis. (Ann. Biologie Clinique, 57(6):643-647 (1999) [French]). Blessum et al. do not disclose the simultaneous isolation of nucleic acids and proteins using isotachophoresis. Dolnik, V. et al. disclose capillary electrophoresis techniques and microchip technology which includes isotachophoresis, and current methods of separation of nucleic acids and proteins, but doe not disclose simultaneous separation of nucleic acids and proteins (Electrophoresis, 21(1):41-54, (1999) [Abstract only]).
U.S. Pat. No. 5,817,225 (Hinton) is drawn to an electrophoresis unit comprising an anode compartment, a cathode compartment, a separation chamber and an electrolyte in said chamber with an electrophoretic mobility between the mobilities of the nucleic acids and organic salts which are to be separated. The separation of proteins was not disclosed.
U.S. Pat. Nos. 7,316,771 B2 and 7,247,224 (withdrawn) (both by Gerhard Weber) is directed to a medium for electrophoresis comprising at least two acids and at least two bases as buffers. Neither disclose the separation of nucleic acids or proteins. U.S. Pat. No. 6,780,584 B1 (Edman et al.) is broadly drawn to a device with a first buffer reservoir containing a first buffer with a differing conductances, a conductive semipermeable matrix, and a first and second electrode and a specific binding entity and discloses a device which separates NUCLEIC ACID and RNA in hybridization reactions, but not the separation of proteins. U.S. Pat. No. 5,464,515 (Bellon) is drawn to a procedure for loading one of several biological samples on an electrophoresis slab support, but does not disclose the separation of nucleic acids.
Newer, rapid, simpler methods of sample processing are required to support the next generation detection systems. The newer detection methods will have a heavier reliance on sample preparation for the generation of meaningful results. In addition, sample preparation methods will need to support numerous different detection systems and be capable of being integrated as part of a complete sample processing and detection system.
Therefore, there is currently a great need in sample processing methods that are fast, inexpensive, and are easy to perform, even by untrained, non-technical staff in a variety of disciplines including biodefense, food and water, agricultural, environmental, clinical testing, and the like.